Immunological assays are designed to detect reactions between antibodies and antigens. These assays commonly employ cells, such as red blood cells (RBCs) or beads as “antigen carriers.” In the appropriate assay configuration, antibodies can cross-link the antigen carriers, generating a large three-dimensional antigen-antibody aggregate from what were initially individual antigen carriers and antibodies. In other configurations, antibodies bind to the antigen carriers without cross-linking them.
Immunohematology testing in the blood bank setting uses RBCs and antibodies to determine compatibility between transfusion donor and recipient prior to transfusion. For example, the donor and recipient are incompatible if antibodies from the recipient cross-link (agglutinate) RBCs from the donor, resulting in the formation of large RBC aggregates. Current commercially available testing reagents are designed to distinguish these aggregates from individual, non-agglutinated RBCs. For example, in standard “tube testing,” RBCs are mixed with antibodies, centrifuged at approximately 1000× acceleration of gravity (g) for a brief period, approximately 30 seconds, to enhance the formation of antigen-antibody complexes, and then gently resuspended by hand in order to be able to distinguish agglutinated from non-agglutinated RBCs. Tube testing is labor-intensive, not amenable to automation, and the results are difficult to standardize from lab to lab since they depend on the skill of the individual operator.
An alternative approach used to identify agglutinated RBCs is spin column technology, which is based on standard chromatographic principles. With this methodology, tubes filled with a homogeneous matrix material, e.g., beads, gel, or polyacrylamide, are used to separate aggregated from individual RBCs. The matrix material is designed with holes or pores of a specified size such that under carefully controlled centrifugal forces large (“4+”) aggregates barely enter the matrix. However, successively smaller aggregates (“3+” through “1+”) do enter the matrix to increasing degrees, and non-agglutinated RBCs not only enter the matrix, but sediment completely to the bottom of the tube. In order for a single homogeneous chromatographic matrix to effectively separate individual RBCs from RBC aggregates of various sizes, a relatively long centrifugation run, approximately 10 minutes, must be carried out under carefully controlled low-speed centrifugation conditions of 80×g. Deviations from optimal centrifugation conditions, e.g., higher centrifugation speeds in an attempt to shorten the assay run, lead to poor separation of RBCs, compromising the assay ability to determine compatibility between blood donor and recipient. This methodology is to some extent amenable to automation, and less dependent on operator skill.
Spin column technology is significantly more expensive than tube testing, due to costs of producing the columns. The matrix material is in solution, and carefully controlled packaging, shipping, and storage conditions are typically necessary. In addition, testing is slower than with tube testing because of the prolonged centrifugation step, approximately 10 minutes, versus approximately 30 seconds with tube testing. Interpretation of assay results also requires operator training, since the readout is on an “analog” scale, i.e., the distance of RBC migration through the matrix must typically be estimated.
There are three main applications of this technology to immunohematology testing: forward-blood typing, reverse-blood typing, and antibody screening. Each of these will be discussed separately.
ABO/D Forward Typing
Forward typing is used to determine the presence of specific clinically-important antigens on the RBC surface. These include, but are not limited to, A-antigen, B-antigen, Rh(D)-antigen, and other RBC antigens including Kell, Duffy, etc. Usually, each of these antigens is tested for in an individual test/reaction. Thus, three separate reactions are required to identify these three RBC antigens. This protocol has conventionally required that three separate tubes/reactions be set up to detect the presence of A, B, and Rh(D) antigens on RBCs.
For A and B antigen typing, we currently use a primary mouse antibody directed against the appropriate antigen, although human antisera can also be used. In theory, these antibodies can be directly labeled with a fluorescent dye, such as fluorescein or any of a number of other commercially available dyes, provided that the analysis device, i.e., flow cytometer or other appropriate instrument, can detect them. Because A and B antigens are composed in part of sugar residues, however, most antibodies that have been prepared against these antigens are of the immunoglobulin M (IgM) class and are difficult to directly label.
IgM anti-A and anti-B antibodies have a propensity to agglutinate RBCs, which is the basis for most commercially available technologies to perform blood typing. However, RBC agglutination prevents the cells from being analyzed by flow cytometry, since the large agglutinates cannot pass through the flow cell but rather clog the flow cell requiring subsequent equipment maintenance. Thus, RBC agglutination has traditionally not been compatible with flow cytometry. Indeed, prior publications in the field have suggested that agglutination of RBCs by antibodies in fact limits the application of flow cytometry to immunohematology. Furthermore, practitioners in flow cytometry usually seek to remove aggregates/agglutinates from the samples prior to flow cytometry so as not to clog the device (e.g., Berneman, Z. N., D. R. van Bockstaele, W. M. Uyttenbroeck, C. Van Zaelen, J. Cole-Dergent, L. Muylle, and M. E. Peetermans, “Flow-Cytometric Analysis of Erythrocytic Blood Group A Antigen Density Profile,” Vox Sang 61:265 (1991); Garratty, G., and P. A. Arndt, “Applications of Flow Cytofluorometry to Red Blood Cell Immunology,” Cytometry 38:259 (1999); Sharon, R., and E. Fibach, “Quantitative Flow Cytometric Analysis of ABO Red Cell Antigens,” Cytometry 12:545 (1991).
ABO Reverse Typing
Reverse typing is used to determine the presence of naturally occurring anti-A and anti-B antibodies in plasma or serum. This test serves as confirmation of the forward typing tests described above to assure that the correct blood type is assigned to an individual. Usually, each of these two antibodies is tested for in individual tests/reactions. Typically, three separate tubes are used that contain group A, B, or O reagent cells. For each tube, an individual's plasma is added, incubated, washed, and then a commercially available fluorescently labeled secondary antibody directed against human IgM is added.
As with forward typing, the presence of IgM anti-A and anti-B antibodies (in this case of human origin) has a propensity to agglutinate RBCs, which is the basis for most commercially available technologies to perform blood typing. Furthermore, as above, RBC agglutination prevents the cells from being analyzed by flow cytometry, since the large agglutinates cannot pass through the flow cell but rather clog the flow cell requiring subsequent equipment maintenance.
Screening for Unexpected RBC Alloantibodies
In individuals, patients, or blood donors that have either been previously transfused or are pregnant, antibodies may have been produced against foreign RBCs (RBC alloantibodies). The same problems apply as for the forward-typing and back-typing assays, including RBC agglutination when strong alloantibodies are present.
Thus, a heretofore unaddressed need exists in the industry to address the aforementioned deficiencies and inadequacies.